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BOR - Papers in Press, published online ahead of print January 25, 2006.
Biol Reprod 2006, 10.1095/biolreprod.105.047803
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BIOLOGY OF REPRODUCTION 74, 865–873 (2006)
DOI: 10.1095/biolreprod.105.047803
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

Injection of Somatic Cell Cytoplasm into Oocytes Before Intracytoplasmic Sperm Injection Impairs Full-Term Development and Increases Placental Weight in Mice1

Nguyen Van Thuan 2 3, Sayaka Wakayama 4, Satoshi Kishigami 3, Hiroshi Ohta 3, Takafusa Hikichi 3, Eiji Mizutani 5, Hong-Thuy Bui 3, and Teruhiko Wakayama 3

RIKEN Kobe Institute, Center for Developmental Biology,3 Laboratory for Genomic Reprogramming, Kobe City, Hyogo 650-0047, Japan Graduate School of Science and Technology,4 Kobe University, Kobe, Hyogo 650-0047, Japan Graduate School of Science and Technology,5 Tohoku University, Tohoku, Japan

ABSTRACT

This study investigated the effects on fertilized embryo development of somatic cytoplasm after its injection into intact mouse oocytes. Mature oocytes collected from female B6D2F1 mice were injected with cumulus cell cytoplasm of different volumes and from different mouse strains (B6D2F1, ICR, and C57BL/6), or with embryonic cytoplasm. After culture for 1 h, B6D2F1 sperm were injected into those oocytes by intracytoplasmic sperm injection (ICSI). The oocytes were examined for pre- and postimplantation developmental competence. Increases in the volume of the somatic cytoplasm from onefold to fourfold resulted in an impairment of blastocyst development and full-term development (28% and 7%, respectively, vs. 96% and 63%, respectively, in the control group; P < 0.01). An increase in the volume of somatic cytoplasm reduced the expression of POU5F1 (more commonly known as OCT4) in expanded blastocysts. The frequency of embryos that developed to the blastocyst stage did not differ when B6D2F1 or ICR somatic cytoplasm was injected, but injection of C57BL/6 somatic cytoplasm induced a two-cell block in embryo development. Injection of the cytoplasm from fertilized embryos did not reduce the frequency of embryos attaining full-term development. Interestingly, somatic cytoplasm significantly increased the placental weight of ICSI embryos, even the injection of onefold cytoplasm (0.20 ± 0.02 [n = 32] vs. 0.12 ± 0.02 in the control group [n = 87]; P < 0.01). These findings indicate that the injection of somatic cytoplasm into oocytes before ICSI causes a decrease in preimplantation development, clearly impairs full-term development, and causes placental overgrowth in fertilized embryos. To our knowledge, placental overgrowth phenotypes are only caused by interspecies hybridization and cloning, and in genetically modified mice. Here, we report for the first time that somatic cytoplasm causes abnormal placentas in fertilized embryos. This study suggests that somatic cell cytoplasmic material is one cause of the low rate of full-term development in cloned mammals.

cumulus cells, developmental biology, early development, embryo, full-term development, ICSI, placenta, preimplantation, somatic cytoplasm

INTRODUCTION

Although cloning mammals by somatic nuclear transfer into oocyte cytoplasts has been performed successfully for nearly a decade, only a very small percentage of cloned embryos have developed to term, with a high incidence of developmental anomalies. These abnormalities include phenotypes such as enlarged placenta, obesity, umbilical hernia, and neonatal death [1–4]. A number of studies analyzing epigenetic modifications during pre- and postimplantation in cloned animals indicate that these cloning failures and abnormalities may be explained by the incomplete reprogramming of the donor cell following its introduction into the oocyte cytoplast [5–9]. These irregularities include abnormal spindle morphogenesis and chromosome distribution during the first cell cycle [10, 11], aberrant epigenetic reprogramming [8, 12, 13], imprinting disruptions [14, 15], X chromosome reaction during development of cloned embryo [16], and abnormal expression patterns of OCT4 (also known as POU5F1), a protein essential for embryonic cell pluripotency and subsequent development [6]. The underlying causes of this inappropriate epigenetic reprogramming, these developmental anomalies, and this failure of full-term development in cloned animals, however, remain unknown.

Many steps are necessary to produce a cloned mammal, in a process that includes the removal of the maternal chromosomes, the injection or fusion of somatic nuclei into the enucleated oocyte, the reconstruction of the somatic chromosomes, oocyte activation, and treatment with cytochalasin B (CB) to produce a diploid cloned embryo. Many of these steps result in a decrease in the developmental rate of blastocysts and high levels of abnormalities in offspring. Moreover, during the process of somatic cell nuclear transfer, all the cytoplasm is introduced into the enucleated oocytes with the donor nucleus, in addition to the genomic material, regardless of whether the electrofusion method is used [17] or the somatic nucleus is injected directly with the Honolulu method (only the donor cell membrane is broken down by microinjection pipetting but the cytoplasm is not removed) [18]. However, there have been no reports demonstrating that somatic cell cytoplasm is a cause of the abnormalities that occur in the development of cloned embryos or in full-term development.

To address this issue, we investigated the effects of somatic cytoplasm from different mouse strains and cytoplasm from fertilized embryos at different stages by injecting them into intact mouse oocytes before intracytoplasmic sperm injection (ICSI). The results of this study indirectly answer the question of whether donor cell cytoplasm may contribute to cloning inefficiency.

MATERIALS AND METHODS

Animals

B6D2F1, ICR, and C57BL/6 mice (Japan SCL, Inc.) were used as sources of oocytes, spermatozoa, and cumulus cells. The ICR strain was used as recipient surrogate mothers for embryo transplantation. All animals were maintained in accordance with the Animal Experimental Hand Book at the Center for Developmental Biology, Riken-Kobe, Japan.

Collection of Oocytes, Cytoplasm, and Spermatozoa

Mature oocytes were collected 14–16 h after female B6D2F1 mice were injected with human chorionic gonadotropin. Somatic cell cytoplasm was collected from the cumulus cells of female B6D2F1, ICR, and C57BL/6 mice after they were removed from mature oocyte-cumulus cells complex by treatment with 0.1% hyaluronidase (Sigma Chemical Co.) in drops of Hepes-buffered Chatot, Ziomek, and Banister (CZB) medium (Hepes-CZB) [19]. To examine the distribution of somatic cytoplasm during preimplantation development after its injection into oocytes before ICSI, some cumulus cells were stained with 20–1000 nM MitoTracker Green FM (Molecular Probes, Inc.) in Hepes-CZB for 5 min [20] and then transferred to a droplet of polyvinylpyrrolidone (Mr 360 kDa; Wako) for cytoplasm collection. After removal of cell membrane, the somatic cell cytoplasm was separated from the cumulus cells with a microinjection pipette of 2–4 µm in diameter and piezo pulses (Fig. 1, A–C1). Embryonic cell cytoplast was collected from B6D2F1 oocytes, B6D2F2 zygotes, and two- and four-cell embryos produced by ICSI. Embryonic cytoplasts were collected using a micromanipulator system and a micropipette with a diameter of 7–8 µm in a droplet of Hepes-CZB with 5 µg/ml CB under mineral oil (Fig. 1, D–D3). Spermatozoa were collected from the two epididymides of mature male B6D2F1 mice and incubated for 30 min at 37°C in 5% CO2 before ICSI.


Figure 1
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  		FIG. 1. Collection of cytoplasm from cumulus cells and embryonic cells by micropipetting and Piezo action and injection into oocytes before ICSI. Cumulus cells were stained with 1 µg/mL bis-benzimide and 20–1000 nM MitoTracker before (A–A2) or after (B–B2) the cytoplasm was detached from the nucleus. Cumulus chromosomes were completely removed from the cumulus cytoplasm with a small pipette and piezo action, and one piece of the cytoplasm collected from one cumulus cell was considered to be onefold (C–C1). Embryonic cytoplasm was removed from the embryonic cell (D–D2) and injected into the oocyte before the zona pellucida was pierced by Piezo action (D3). Injection of onefold, twofold, threefold, and fourfold cumulus cytoplasm into oocytes (E–E3) and different volumes of mitochondrial DNA were observed after injection of different volumes of somatic cytoplasm (F–F1). Distribution of the mitochondrial DNA from the somatic cytoplasm from a two-cell fertilized embryo (G–G1). Nuclear DNA is shown in blue and mitochondrial DNA of somatic cytoplasm in green. Bar = 50 µm.

Injection of Somatic Cell Cytoplasm, Embryonic Cell Cytoplasm, and Spermatozoa

After collection, the collected somatic cell cytoplasms were incubated in Hepes-CZB droplets containing 1 µg/ml Hoechst 33342 to confirm the absence of chromosomes under ultraviolet light (Fig. 1, C–C1). Cytoplasm with no chromosomes was injected into oocytes in volumes of onefold, twofold, threefold, and fourfold (Fig. 1E–E3). A onefold cytoplasm is equal to one fourth the volume of one entire cumulus cell cytoplasm, and a fourfold volume of cumulus cytoplasm is equivalent to the volume of one entire cumulus cell cytoplasm (Fig. 1C–C1). The volume of embryonic cytoplasts was equivalent to double the fourfold volume of the cumulus cytoplasm (eightfold). The volumes of cumulus cytoplasm and embryonic cytoplasts were measured visually under a manipulator microscope. Collected embryonic cell cytoplasts was washed three times in Hepes-CZB and then transferred into a droplet of the same medium for injection. The membrane of the embryonic cell cytoplast was removed during injection. The cytoplasm was injected into an oocyte using an injection pipette of 7–8 µm diameter and piezo pulses, with a manipulator system. After cytoplasm injection, the oocytes were cultured in KSOM/AA (Specialty Media) containing 1% BSA (KSOM medium) for 1 h (equivalent to the time required for donor chromosomes to achieve a metaphase-like structure after injection into the enucleated oocyte and before activation) [1, 11]. B6D2F1 sperm were injected into the B6D2F1 oocytes by ICSI; then, the fertilized cells were cultured in KSOM medium in an atmosphere of 5% CO2 and examined for preimplantation developmental competence. ICSI was performed with the previously reported method of Kimura and Yanagimachi [21], described in detail by Van Thuan et al. [22].

Nuclear Staining and Differential Staining of Trophectoderm Cells and Inner Cell Masses in Expanded Blastocysts

Expanded blastocysts were treated with acid Tyrode solution for 1–2 min to remove the zona pellucida, and washed twice in Ca2+-free, Mg2+-free Dulbecco PBS containing 0.1% polyvinyl alcohol (PBS-PVA; Sigma), then fixed for 30 min in PBS-PVA with 3.5% paraformaldehyde. The fixed blastocysts were washed twice in PBS-PVA and stored overnight at 4°C in PBS supplemented with 3% BSA (PBS-BSA; Sigma) and 0.1% Triton X-100 (Nacalai Tesque Inc.). To stain the trophectoderm (TE) cells and inner cell masses (ICM), the samples were incubated for 90 min at room temperature in mouse anti-CDX2 IgG (1:100 dilution; BioGenex), a TE marker, and rabbit anti-OCT4 IgG (POU domain, class 5, transcription factor 1, also known as OCT3/4, 1:100 dilution; Santa Cruz Biotechnology), an ICM marker. After the samples were washed twice in PBS-BSA, they were incubated with Alexa-Fluor-568-labeled goat anti-mouse IgG and Alexa-Fluor-488-labeled chicken anti-rabbit IgG antibodies (1:100 dilution; Molecular Probes). After three 10-min washes in PBS-BSA, the DNA was stained for 30 min with 2 µg/ml 4',6-diamidino-2-phenylindole dihydrochloride (Molecular Probes). After the samples were washed thoroughly, they were mounted on slides using Vectashield mounting medium (Vector Laboratories Inc.) and observed with a Bio-Rad Radiance 2100 confocal scanning laser microscope (Bio-Rad). The process of fixation and the staining of samples were performed in 96-well culture dishes (Scienceproducts), and the samples were transferred to subsequent treatments by mouth pipette.

Culture for Development and Embryo Transfer

In each experiment, spermatozoa-injected oocytes were cultured in droplets of KSOM medium under paraffin oil in plastic dishes at 37°C in a 5% CO2 incubator. The stages of embryonic development were evaluated under an inverted microscope at 24-h intervals. Embryo transfer was performed 24 h (two-cell embryo) or 72 h (morula and blastocyst stage) after ICSI; 10–15 two-cell embryos or morulae and blastocysts were transferred into the oviducts or uterus of each surrogate mother (ICR mouse) on Day 1 or Day 3 of pseudopregnancy, respectively, following mating with vasectomized ICR males. To examine the effects of fetus number on body and placental weights, only two two-cell embryos were transferred to some surrogate mothers in the control group (sham injection before ICSI), whereas 10–15 two-cell embryos were transferred to other surrogate mothers.

Statistical Analysis

Each experiment was repeated four to five times to obtain 80–100 oocytes per treatment. The data were subjected to arcsine transformation for each replication. The transformed values were analyzed using one-way ANOVA. Values of P < 0.05 were deemed to indicate statistical significance.

RESULTS

Distribution of Somatic Cytoplasm after Injection into Oocytes Followed by ICSI

Immediately after its injection, the somatic cytoplasm was located at the injection site (Fig. 1, F–F1). One hour after ICSI, the somatic cytoplasm was dispersed into the oocyte cytoplast, and it had spread throughout the embryo 6 h after ICSI. At the two-cell embryo stage, the somatic cytoplasm was distributed in both embryonic cells (Fig. 1, G–G1). Stained somatic cytoplasm was observed in all embryonic cells at the four-cell stage. However, at the morula and blastocyst stage, the green color of MitoTracker was not observed. When the concentration of MitoTracker was increased to 1000 nM and examined once after ICSI at the expanded blastocyst stage, we observed the green color of MitoTracker in most blastomeres. These results suggest that somatic cytoplasmic materials are active following their injection into the recipient oocyte and during the preimplantation development of cloned embryos.

Effects of the Volume of Somatic Cell Cytoplasm and Embryonic Cytoplasm on the Preimplantation Development of the Fertilized Embryo

No difference was observed at the two-cell stage when the volume of B6D2F1 cumulus cytoplasm was increased from onefold to fourfold (Table 1). At 48 h after ICSI, the frequency of surviving oocytes injected with fourfold somatic cytoplasm was significantly lower than the frequency of surviving oocytes injected with onefold somatic cytoplasm or those in the control group (78% vs. 93% and 99%, respectively; P < 0.05). However, at 72 h after ICSI, the frequencies of embryos that had developed to the morula and blastocyst stage were significantly lower in the group in which oocytes were injected with fourfold somatic cytoplasm than in the groups injected with onefold to twofold somatic cytoplasm or in the control group (P < 0.05, Table 1). There was no significant difference between the group injected with onefold cytoplasm and the control group. On the other hand, when oocytes were injected with cytoplasm from B6D2F1 oocytes, B6D2F2 zygotes, or two- or four-cell embryos before ICSI, the frequencies of oocytes that became zygotes were low (Table 2). However, there was no significant difference between the experimental groups and the control group during the development from two-cell embryos to blastocysts or full-term neonates (Table 2).


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  		TABLE 1. Effects of the volume of somatic cell cytoplasm on the preimplantation development of the fertilized embryos.


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  		TABLE 2. Effects on the preimplantation development of fertilized embryos and full-term offspring of oocyte and embryonic cytoplasm.

Effects of Somatic Cell Cytoplasm from Different Mouse Strains on the Preimplantation Development of the Fertilized Embryos

In this experiment, onefold and fourfold volumes of somatic cytoplasm from different mouse strains were injected into B6D2F1 oocytes 1 h before ICSI. The results are shown in Table 3 and Figure 2. The frequencies of embryos that developed to the four-cell or morula and blastocyst stages at 48 and 72 h after ICSI, respectively, were usually low after the injection of fourfold somatic cytoplasm, compared with those after the injection of onefold somatic cytoplasm, regardless of the mouse strain (P < 0.05; Table 3 and Fig. 2). There was no significant difference between embryos derived from the B6D2F1 and ICR donor mouse strains at the four-cell stage after the same volume of cumulus cytoplasm was injected. However, only 54% of oocytes injected with C57BL/6 somatic cytoplasm developed to four-cell embryos. Interestingly, we observed that more than 30% of these embryos were blocked at the two-cell stage in the C57BL/6 group (Fig. 2C), and the phenomenon of this two-cell block correlated directly with the volume of somatic cytoplasm injected. In the groups of oocytes injected with onefold and fourfold C57BL/6 somatic cytoplasm, only 39% and 22% of fertilized embryos, respectively, developed to the morula and blastocyst stage (vs. 90% and 64%, respectively, in the B6D2F1 group, and 87% and 58%, respectively, in the ICR group; P < 0.05). However, all the oocytes injected with fourfold somatic cell cytoplasm produced low frequencies of morulae and blastocysts compared with the controls, regardless of the mouse strain.


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  		TABLE 3. Effects on the preimplantation development of ICSI mouse embryos.


Figure 2
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  		FIG. 2. Fertilized mouse morulae and blastocysts derived from oocytes injected with fourfold cumulus cytoplasm from various mouse strains before ICSI. Oocytes were injected with B6D2F1 cumulus cytoplasm (A), ICR cumulus cytoplasm (B), or C57BL6 cumulus cytoplasm (C), and their developmental competence was observed 72 h (morula and early blastocyst stage) after ICSI. Control group contained oocytes sham-injected with no somatic cytoplasm before ICSI at the blastocyst stage (D). Asterisk indicates the two-cell block when injected with C57BL6 cumulus cytoplasm (C). Bar = 100 µm.

Effects of Somatic Cytoplasm on the Number of Blastomeres and the Expression of OCT4 at the Expanded Blastocyst Stage

CDX2 and OCT4 have been identified as specific markers for the TE and ICM, respectively, at the expanded blastocyst stage [23, 24]. To examine whether the somatic cytoplasm of different mouse strains affects the qualities of fertilized blastocysts, such as total cell number, the numbers of TE and ICM cells were examined with CDX2 and OCT4 markers, respectively. There was no significant difference in the numbers of blastomeres expressing CDX2 in the expanded blastocysts (Fig. 3A). However, the average numbers of blastomeres expressing OCT4 were low in all groups in which the oocytes were injected with somatic cytoplasm before ICSI compared with the control group, regardless of the mouse strain. This was especially marked in those groups of oocytes injected with fourfold somatic cytoplasm. The total numbers of cells and the numbers of blastomeres expressing OCT4 were lowest when oocytes were injected with C57BL/6 somatic cytoplasm. Although there was no significant difference between the total number of cells in the control group and in the groups injected with onefold B6D2F1 or ICR cumulus cytoplasm (P > 0.05; Fig. 3A), the number of OCT4-positive blastomeres was significantly reduced (P < 0.05; Fig. 3A). These results indicate that some ICM cells did not express OCT4. Therefore, a small volume of donor cell cytoplasm did not affect the cell numbers of the expanded blastocysts but did inhibit the expression of OCT4 in ICM cells. To examine the correlation between somatic cytoplasm and the expression of OCT4 in expanded blastocysts, we categorized the expanded blastocysts into four classes based on the numbers of OCT4-positive ICM cells (I, n > 15; II, 10 < n ≤ 15; III, 5 < n ≤ 10; and IV, 0 < n ≤ 5; Fig. 3B). Nearly 80% of the expanded blastocysts in the control group (without somatic cytoplasm injection) contained more than 15 OCT4-positive blastomeres. However, the numbers of OCT4-positive blastomeres were reduced in those groups in which oocytes were injected with onefold or fourfold somatic cytoplasm (Fig. 3C). This result clearly indicates that the injection of somatic cytoplasm into oocytes before ICSI resulted in low-quality expanded blastocysts, with a decrease in OCT4 expression in ICM cells.


Figure 3
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  		FIG. 3. Total blastomeres and OCT4 and CDX2 expression in expanded blastocysts derived from oocytes injected with onefold or fourfold cumulus cytoplasm from different mouse strains before ICSI, and the control group. A) The number of blastomeres showing CDX2 or OCT4 expression in expanded blastocysts derived from B6D2F1 oocytes injected with onefold or fourfold cumulus cytoplasm from B6D2F1 (F1 x 1 and F1 x 4), ICR (ICR x 1 and ICR x 4), or C57BL/6 (B6 x 1 and B6 x 4) mouse strains before ICSI, and the control group. B and C) Classification of expanded blastocysts based on the numbers of blastomeres expressing OCT4. Control group contains oocytes sham-injected with no somatic cytoplasm before ICSI. CDX2 is shown in red and OCT4 in green. Bars represent means ± SD. Different letters indicate significantly different values (P < 0.05). Bar = 50 µm.

Effects of Somatic Cytoplasm on the Full-Term Development and Placental Weight of the Fertilized Embryos

The effects of the somatic cytoplasms of different mouse strains on full-term development and placental weight are shown in Table 4. The birth rates of fertilized embryos that had been injected with somatic cytoplasm before ICSI were significantly decreased and were dependent on the volume of somatic cytoplasm injected but not on the donor mouse strain. Only 6.3%, 8.7%, and 7.4% of fertilized embryos developed to term after the injection of fourfold volumes (equivalent to the volumetric cytoplasm of one cumulus cell) of somatic cytoplasm from B6D2F1, ICR, and C57BL/6 mice, respectively. The full-term development frequencies in the group 1 controls (transfer of 10–15 embryos/surrogate mother) and group 2 controls (transfer of two embryos/surrogate mother) were 57% and 38%, respectively. We also achieved a development frequency of 1.7% cloned mice when cumulus cell nuclei were injected into enucleated oocytes, followed by activation and CB treatment. These observations show that this impairment of full-term development is caused by the injection of somatic cytoplasm but is not affected by the number of embryos transferred. However, analysis of the correlation between the number of embryos transferred per surrogate mother, the number of offspring, and their body weights shows that the body weight of offspring increases when the frequency of full-term offspring decreases. The body weights of pups derived from oocytes injected with onefold somatic cytoplasm were always lower than those of pups injected with fourfold somatic cytoplasm (P < 0.05; Table 4). This result was confirmed when two fertilized embryos were transferred to each surrogate mother; the body weights of the offspring were similar to those of cloned mice but higher than those of pups derived from the transfer of 10–15 embryos per surrogate mother (P < 0.01; Table 4). We also found that the offspring of cloned mice were heavier than those of the group 1 controls or those of the onefold and fourfold somatic cytoplasm groups, but there was a significant difference between the weights of the fourfold somatic cytoplasm group and the group 2 controls. Although there is no direct proof, when the numbers of OCT4-positive ICM cells in the expanded blastocysts (Fig. 3) and the frequency of full-term development (Table 4) are compared, it is clear that the expanded blastocysts in group A are mainly those that develop to term. This may indicate that only expanded blastocysts containing more than 15 OCT4-positive ICM cells are competent to pass through the postimplantation process and develop to term. Interestingly, the placental weights of the groups in which the somatic cytoplasm was injected into oocytes before ICSI were always higher than those of the control groups, even in those in control group 2, in which two embryos were transferred into each surrogate mother (P < 0.05; Table 4, Fig. 4A–C). When we examined the effects of oocyte or embryonic cytoplasm on placental weight, there was no significant difference in placental weights between the experimental groups and the control group (B6D2F1 oocytes, 0.10 ± 0.03; B6D2F2 zygotes, 0.12 ± 0.02; B6D2F2 two-cell, 0.10 ± 0.02; B6D2F2 four-cell, 0.11 ± 0.01; vs. control, 0.11 ± 0.03, P > 0.05). Interestingly, the placental weights in the groups injected with somatic cytoplasm were higher than those of the control groups, but they were significantly lower than the placental weights of cloned mice (Table 4, Fig. 4D). Surprisingly, we recovered only placenta without fetus when oocytes were injected with fourfold B6D2F1 cumulus cytoplasm before ICSI (Fig. 4B). This phenomenon has often been observed in cloned mice [25] but never in normal fertilized embryos. Moreover, some fetal deaths with large placentas were observed in the somatic-cytoplasm-injected groups (1% in onefold and 4% in fourfold, Fig. 4A).


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  		TABLE 4. Somatic cytoplasm volume: Effects on the full-term development and body and placental weights of newborn mice from different mouse strains injected into B6D2F1 oocytes before ICSI.


Figure 4
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  		FIG. 4. Normal and abnormal full-term development in mice. A) Dead fetus derived from an oocyte injected with somatic cytoplasm before ICSI, showing an enlarged placenta and umbilical hernia at 19.5 days postconception. B) A pup with an overgrown body and placental hypertrophy, and a placenta with no fetus, were derived from a single surrogate mother after oocytes were injected with fourfold somatic cytoplasm before ICSI. C) A pup from the control group in which 10–15 embryos were transferred per surrogate mother, showing normal body and placental weights. D) A pup derived from a somatic nuclear transfer embryo showing bodily overgrowth and placental hypertrophy.

DISCUSSION

The first successfully cloned mammal [17] was generated nearly a decade ago. Since then, several mammals, including mice [18], cows [26], goats [27], pigs [28], rabbits [29], cats [30], a mule [31] horses [32], rats [33], and recently dogs [34], have been cloned by the transfer of a somatic cell nucleus into an oocyte from which the chromosomes have been removed. Many researchers have endeavored to increase the developmental competence and decrease the typical malformations seen in somatically cloned embryos. For example, they have changed the activation timing and the fusion/activation conditions [35–37] or the culture conditions of the donor cells and cloned embryos [38–41]; treated them with dimethyl sulfoxide [36], trichostatin A, or 5-aza-2'-deoxycytidine [40, 41]; altered the timing of the removal of the oocyte chromosomes [42]; and introduced clone-clone aggregation [43] and serial nuclear transfer [44]. However, the success rate in cloning animal offspring has remained very low. These offspring have manifested many abnormal phenotypes throughout preimplantation to the birth of the offspring, including low numbers at the blastocyst stage with abnormal epigenetic expression, fetal death during postimplantation, placental edemal and umbilical hernia at birth, or obese phenotype at maturity in mice [1–4]. Theoretically, in nuclear transfer only the genetic material of the somatic cell is transferred into the mature oocyte cytoplast, which contains no cytoplasm. However, in reality, the entire somatic cell cytoplasm (equivalent to a fourfold volume in this study) is introduced into the oocyte cytoplast, regardless of whether the fusion method or the direct injection method is used. The somatic cell is a differentiated cell. Therefore, somatic cytoplasm contains proteins and materials that control the cell's differentiation pathway. However, the characteristics of the somatic cytoplasm that affect the cloned embryo remain unclear. For that reason, the aim of this study was to define the effects of somatic cytoplasm, after its injection into the oocyte and before ICSI, on the development of the embryo and at term.

With the data presented here, we clearly show that the injection of somatic cytoplasm into oocytes before ICSI causes a decrease in their preimplantation development and clearly impairs the full-term development of fertilized embryos, even when the same mouse strain is used as the somatic cytoplasm donor and recipient oocyte. The effects of somatic cytoplasm on the development of fertilized embryos are dose dependent. Even the injection of only onefold cytoplasm (1/4 of the cytoplasm of one cumulus cell) reduces by more than half the proportion of fertilized embryos that develop to term. When the volume of somatic cytoplasm is increased to equal the volumetric cytoplasm of one cumulus cell (fourfold), full-term development was severely impaired (1%–9%). The cytoplasm of oocytes or of one-cell to four-cell embryos did not affect full-term development after it was injected into oocytes, even at volumes double that of fourfold somatic cytoplasm. These results indicate that the cytoplasm of differentiated cells (cumulus cells), but not that of undifferentiated cells (oocytes and one- to four-cell embryos), is harmful to fertilized embryos during pre- and postimplantation development. Interestingly, the presence of somatic cell cytoplasm in fertilized embryos caused abnormal phenomena in the offspring that were also observed in cloned embryos, such as decreased OCT4 expression in ICM cells at the expanded blastocyst stage, increased placental overgrowth in fertilized embryos, development of the placenta only, and dead fetuses with large placentas. In the mouse, placental overgrowth has only been seen previously with interspecific hybridization [45], with the deletion of some genes such as Esx1 [46] or Lpl [47], and in mice cloned from somatic cell nuclei [1–3, 48]. Of those, only cloned mice generate placental overgrowth without the contribution of genetic factors [49].

Although the injection of the cytoplasm of B6D2F1 oocytes or B6D2F2 one-, two-, four-, or eight-cell fertilized embryos into B6D2F1 oocytes before ICSI caused a decrease in the number of two-cell embryos, no effect was observed on their later development and placental weight was normal. This decrease in the number of two-cell-stage embryos after the injection of embryonic cytoplasm occurs because the membrane of embryonic cytoplast is broken down during the process of injection. Therefore, some embryonic cytoplasm is shattered under the influence of piezo action during the injection process, which induces the degeneration of the recipient oocyte. Therefore, the pronucleus cannot form and the sperm-injected oocytes degenerate. However, most of them can develop from the two-cell embryo to the morula and blastocyst stage. This observation supports the evidence that the cytoplasm of somatic cells contains differentiated material, whereas oocyte and embryonic cytoplasm contains undifferentiated factors. Both types of cytoplasm contain the same organelles, such as mitochondria, derived from the female B6D2F1 mice, but only the materials of the somatic cells had a harmful effect on the development of fertilized embryos, whereas the embryonic cytoplasm did not. This suggests that beside mtDNA, there are factors present in the somatic cytoplasm that detrimentally affect the development of the embryo, decrease OCT4 expression in the expanded blastocyst, and cause the overgrowth of the placenta of the cloned animal. This hypothesis is supported by the fact that when the volume of cumulus cell cytoplasm was increased to fourfold (equivalent to the cytoplasm of one cumulus cell), abnormalities also increased, including the high frequency of expanded blastocysts with low OCT4 expression, a phenomenon previously observed in cloned embryos [6, 50]. Our study shows that the failure of OCT4 expression in ICM cells is induced not only by the somatic cell genome but also by somatic cytoplasmic materials. Thus, our observations suggest that donor cell cytoplasm may contribute to cloning inefficiency. We tried to remove the somatic cytoplasm by using a small microinjection pipette, but this may have caused considerable damage to the donor chromatin, and ultimately could not support the full-term development of cloned embryos (data not shown).

On the other hand, overgrowth of the fetus correlates inversely with the number of embryos transferred. In cloned mice, although 15–20 cloned embryos were transferred per surrogate mother, a survival rate of only 0.1%–2% of offspring was achieved [1, 51]. Therefore, the overgrowth of the cloned fetus may correlate with the number of live fetuses or the number of offspring at birth, but is not affected by abnormal reprogramming of the somatic genome. The present study shows that in control group 2 (only two embryos transferred per surrogate mother), the weight of the neonates was significantly increased compared with that of control group 1 (10–15 embryos transferred per surrogate mother), although it was equivalent to that of cloned full-term neonates.

Several studies have demonstrated that the donor cell genotype significantly affects the preimplantation [52] and full-term development of cloned embryos [53]. Inoue et al. [54] reported that donor cell types of the same genotype also affect the success of cloning. These different effects of the donor cell genotype and the donor cell type do not provide a clear indicator of the donor genomic and/or cytoplasmic factors involved in the development of cloned embryos. It is very difficult to separate only the intact chromosomes from the donor cell to examine the effects of the genome on developmental cloning. Therefore, the effects of the donor genome and donor cytoplasmic factors on the development of cloned embryos are still unclear. In this study, our data clearly show that donor cytoplasm significantly affects the preimplantation and full-term development of fertilized embryos. Our results also indicate that donor cytoplasm exerts strain-dependent effects on the development of fertilized embryos. Thus, preimplantation development was impaired and the cloning success rate was very low when the donor nuclei were isolated from the C57BL/6 mouse strain compared with the B6D2F1 strain [52, 53]. We also observed the phenomenon of the two-cell block, which occurred after the injection of somatic cell cytoplasm from the C57BL/6 mouse strain into B6D2F1 oocytes before ICSI. This indicates that not only the oocyte cytoplasm of the C57BL/6 mouse strain [55–57] but also the somatic cytoplasm of the C57BL/6 mouse strain induces the two-cell block phenomenon after it is injected into B6D2F1 mouse oocytes (a non-two-cell-block mouse strain).

In conclusion, the findings of this study demonstrate, for the first time, that the injection of somatic cytoplasm into oocytes before ICSI causes a decrease in preimplantation development and clearly affects the qualities of embryos, such as the total number of cells and OCT4 expression in ICM cells at the blastocyst stage. It ultimately impairs full-term development and increases the placental weight of fertilized mouse embryos. This study also suggests that somatic cell cytoplasmic material is one cause of the low rate of full-term development and the typical malformations seen in somatically cloned mice. Further studies are required to increase the developmental competence of cloned mice by eliminating the somatic cytoplasm, or by treating somatic cells to remove these factors from the cytoplasm before nuclear transfer.

FOOTNOTES

1 Supported by grants-in-aid for Creative Scientific Research (13GS0008) and for Young Scientists (A) (15681014) to T.W. from MEXT, Japan. Back

2 Correspondence: Nguyen Van Thuan, RIKEN Kobe Institute, Center for Developmental Biology, Laboratory for Genomic Reprogramming, 2–2–3 Minatojima-minamimachi, Chuo-ku, Kobe City, Hyogo 650-0047, Japan. FAX: 81 78 306 3095; nvthuan{at}cdb.riken.jp Back

Received: 22 September 2005.

First decision: 14 October 2005.

Accepted: 19 January 2006.

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